Glomerular disease is a leading cause of chronic and end-stage renal disease. Podocytes are highly specialized cells lining the outer aspect of the GBM, and functions thereby prevent protein leakage and also maintain the normal glomerular capillary integrity. In contrast to other renal cells, podocytes replicate to replace those lost due to injury, and studies have shown that decreased podocyte number causes glomerulosclerosis. The focus of this grant is to delineate novel mechanisms that prevent podocytes from proliferating in disease. Proliferation is governed at the level of the cell cycle by positive (cyclins and cyclin-dependent kinases) and negative (cyclin-dependent kinase inhibitors) cell cycle proteins. Recent evidence shows that cell cycle proteins are in turn controlled by other events, such as DNA damage. Cell cycle arrest is necessary in order for the cell to repair any damaged DNA, so as to not propagate this to daughter cells. The goal of this grant proposal is to demonstrate that DNA damage is a novel response of podocytes to immune (sublytic C5b-9) and nonimmune (puromycin aminonucleoside) injury. We will test the central hypothesis that DNA damage reduces podocyte number by preventing proliferation, thereby leading to glomerulosclerosis. In the first specific aim, we will show that injury to podocytes in vitro and in vivo in experimental glomerular disease in rats and mice causes DNA damage. We will elucidate the mechanisms of this effect by testing the hypothesis that sublytic C5b-9 injury causes oxidative stress, and that the membrane attack complex (C5b-9) assembles on the podocyte's nuclear membrane. Because our preliminary data shows that immune-mediated injury does not cause DNA damage in mesangial cells, we will determine the mechanisms underlying this differential effect in glomerular cells. In the second aim, we will determine the consequences of DNA damage on podocyte proliferation and detachment in cell culture and in experimental glomerular disease. We will test the hypothesis that p53 and the CDK-inhibitor p21 mediate the anti-proliferative effects of DNA damage on podocytes, utilizing cultured podocytes derived from p53 and p21 null mice, and by also inducing experimental injury in these mice. Finally, we will also determine the signaling pathways (ERKI/2, JNK, p38) underlying DNA damage, and test the hypothesis that these regulate the levels of p53 and p21 in podocytes. The proposed in vitro and in vivo experiments will delineate novel insights into podocyte biology that are very relevant to disease. The ultimate goal is to identify new targets for potential therapies in order to reduce the heavy burden of disease in patients with gIomerular disease.